Analysis of Plasmodium vivax Apical Membrane Antigen-1 (PvAMA-1) Haplotypes among Iranian Isolates

Plasmodium vivax apical membrane antigen-1(PvAMA-1) is a surface protein with polymorphic sites. This study was aimed to analyze the polymorphic amino acid residues at PvAMA-1 in different infected age groups. 92 blood samples were collected from the south and southeast of Iran. The DNA coding for the domain I (DI), DII, and partial DIII of this antigen was amplified by Nested-PCR, and sequenced. Nucleotide mutations were found in 49 sites and based on the amino acid sequence, 30 variable sites were detected. Age distribution of malaria cases showed that the majority of the patients were between 10 to 30 years old. The scattering plot haplotypes by age showed an increasing incidence rate with age during childhood, whereas, incidence was the lowest in patients under five years old. Comparison of the polymorphic sites of PvAMA-1 in Iranian isolates with those found in other geographic regions of the world indicated nine common variable positions. In addition, a significant dependence was found between some particular substitutions and age categories. Dependence between particular substitutions and age groups suggests that certain residues in AMA-1 are responsible for clinical attacks in different ages, likely as a result of host immune pressure. The crystal structure of the PvAMA-1 showed that the amino acid substitutions that changed the protein charge were exclusively located in loops and turns where, the interactions with antibodies could occur. These data provide the necessary information for an AMA-1 based malaria vaccine design to be effective across all ages.

most prevalent form of malaria outside Africa (3).
Therefore, development of a vaccine against P.
vivax is a necessary priority, particularly due to increased resistance of this parasite to the antimalarial drugs such as chloroquine, primaquine and pyrimethamine (4)(5)(6)(7). Identification of target antigens that induce protective immune response is a pre-requisite for vaccine development. Many reports indicate apical membrane antigen1 (AMA-1) as one of the promising malaria vaccine candidates (8)(9)(10)(11). AMA-1 is a type I integral membrane protein found in most of the Plasmodium species (11). AMA-1 expression is maximal in late schizogony during asexual reproduction in red blood cells (12,13). It is initially located in merozoite apical organelles, and after processing relocates to the surface of mature merozoite (14). Around the time of erythrocyte invasion, the pro-sequence of AMA-1 is processed proteolytically by two c-terminal cleavages (15).  (16). The stage specificity, and location of AMA-1 suggest that this protein is associated with the process of erythrocyte invasion as shown by the inhibition of parasite invasion using antibodies against AMA-1 (17).
Also, generation of the parasites with disrupted ama-1 gene by knockout technology suggests a critical role for AMA-1 in the development of the parasite during asexual blood-stage (18).
Vaccination with AMA-1 can provide protective immunity in mice and monkeys, and induce antibodies that inhibit parasite development in vitro. These evidences introduce AMA-1 as a promising blood-stage vaccine candidate. In addition, unlike most other blood-stage proteins, ama-1 gene, excluding some regions, is a conserved protein among various Plasmodium species. Point mutations are responsible for limited diversity in this antigen, and most of them are in DI (19,20).
Antigenic diversity is supposed to be a major mechanism for the parasite to escape from the host immune system, but remains the greatest obstacle in designing a malaria vaccine. Some findings show that protective immunity against AMA-1 and MSP-1 (merozoite surface protein) has a high degree of strain specificity (22,23). Thus, AMA-1 polymorphism is a major problem to the efficacy of this protein as a vaccine component. On the other hand, some epitopes of this antigen produced by different allelic forms may be involved in the manifestation of clinical symptoms in particular age groups. Such variants should be considered to prepare repertories of AMA-1 allelic forms for developing a universal malaria vaccine. In the current study, the P. vivax ama-1 gene was amplified and sequenced from 92 blood samples collected from two different malaria endemic regions of Iran. The study was conducted to analyze the population structure of pvama-1 Iranian isolates as an important malaria vaccine candidate. Finally, this investigation introduces the common mutation sites that should be considered for vaccine design.  T/A that are also responsible for the generation of new parasite variants (Table 1).

Discussion
The present study was aimed to investigate the associations between the incidence of PvAMA-1 variants and age of infection.
The data show that the incidence of malaria reached the peak in the late teenage years, and declined with age in adulthood ( Table 2; Fig. 1). This age distribution confirms that in populations with low malaria transmission, the incidence increases with age in teenagers and then declines; whereas in areas exposed to a very low level of transmission or to epidemic malaria, the clinical attack occurs across all ages (33). The decrease in malaria intensity with age in adults in the present study is in agreement with the study on P. falciparum in Kwazulu Natalin South Africa (34), and another report on P. vivax infection by Denis-lozano et al. (35) in Mexico that can be explained by the low level of transmission intensity in these areas. Besides, adults may have had immunity early in life due to previous exposure to the malaria parasite. Our findings are in contrast with the report of Baird et al. (36) in Irian Jaya which showed an increase in malaria infection with age in a population of adults from Java after migration to a hyper endemic area. However, the examined population in the present study has been exposed to a lower level of transmission than the population in the Irian Java study. The scattering plot shows an increasing incidence rate with age Domain II (Fig. 2 A and B) which were also found in India, Venezuela and Sri Lanka (Table 3).
Despite this, the Q277K replacement has not been found in Thai population that may be explained by the lower host immune selection pressure due to very low malaria incidence rate (30) and/or insufficient time to generate new alleles bearing this mutation. Moreover, some mutations involved in the amino acid changes with the same charge may be associated with functional constraints of the protein (K120R, D242E in AMA-1 Domain I) (Fig. 2 A).
Comparison of the polymorphic positions between five different populations showed that 9 positions were common ( Table 3). The conservation of the common mutations suggests that these particular substitutions are necessary for the parasite to escape from the host immune system (21). If other mutations could play a similar role in the destruction of epitopes targeted by the immune system, different mutations would probably be expected between separate populations as shown in various haplotype repertoires. The polymorphic amino acid residues of each mutation site in the studied age groups were compared with that of the whole population by the two tailed Fisher exact test in Graph pad program.*: significant values with P<0.05;**: significant values with P< 0.0001 The distribution of polymorphic amino acid residues (common between 5 comparative regions) by age categories (Table 4)  showed that residues E187 and E243 were strongly associated with malaria incidence in children under Concerning the effect of certain amino acid residues on malaria infection rates, we cannot determine if these residues affect directly or their cooperation with other AMA-1 residues involved. yet, as the amino acid residues associated with the clinical malaria cases occur in multiple AMA-1 sequences, few parasite transmission can be ruled out. Thus, it is improbable that the clinical attacks are due to a combination of particular residues in other loci.
Knowledge of the amino acid substitutions, particularly those with charge conversion, could help to recognize the main epitopes that elicit protective antibodies. In the present study, these substitutions were exclusively observed in turn and loop secondary structures of the antigen (Fig. 3).
Turns and loops generally lie on the surface of the proteins where they can participate in interactions between proteins and other molecules such as antibodies (38,39).
The present study provides the necessary information to design a malaria vaccine to be effective in different age categories, and suggests that some of the epitopes in AMA-1 providing protective immunity response are strain specific.
This problem is one of the difficulties in designing efficient vaccine based on only one allelic form of the protein, and was confirmed by previous reports (17,19,22,40)